Rhodopsin in Bovine Rod Outer Segment Disk Membranes Exhibits Two Thermal Transitions

Abstract

The photoreceptor, rhodopsin is a GPCR in rod outer segment disk membranes. Activation by light converts the dark-adapted form, rhodopsin is to the bleached form, opsin. Differential scanning calorimetry (DSC) studies showed that rhodopsin and opsin each exhibit an irreversible scan rate dependent endothermic transition (Tm) at approximately 72oC and 55oC respectively. We report here a scan rate dependent exothermic transition. Solubilization was used to examine the contribution of the bilayer. Freeze-thaw cycles were used to examine rhodopsin aggregation in these transitions. Disk membranes were sequentially solubilized in octylglucoside (OG) until rhodopsin was completely delipidated. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90°/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. As the OG partitioned into the bilayer the endothermic Tm and Eact (activation energy of denaturation) rapidly decreased. Both then remained constant following rhodopsin solubilization. At low detergent concentration the exothermic Tm increased rapidly then remained constant after solubilization. Unlike the endothermic Eact, the degree of solubilization had little effect on the exothermic transition Eact. Freeze-thaw cycles generated disks in which rhodopsin was aggregated. Increasing aggregation resulted in a decrease in the Tm of the exothermic transition, but had no effect on the endothermic Tm of either rhodopsin or opsin. These results are consistent with earlier cross-linking studies suggesting the exothermic transition results from aggregation. These results indicate that the rhodopsin endothermic transition is due to weakening of the tertiary structure interactions accompanied by changes in the packing of the trans-membrane helices as well as changes in protein-lipid interactions. CD data indicate that rhodopsin secondary structure is almost unchanged.